Abstract

Deregulated production of IL-17 and IL-21 plays a key pathogenic role in many autoimmune disorders. A delineation of the mechanisms that underlie the inappropriate synthesis of IL-17 and IL-21 in autoimmune diseases can thus provide important insights into potential therapies for these disorders. Here we have shown that the serine-threonine kinase Rho-associated, coiled-coil–containing protein kinase 2 (ROCK2) becomes activated in mouse T cells under Th17 skewing conditions and phosphorylates interferon regulatory factor 4 (IRF4), a transcription factor that is absolutely required for the production of IL-17 and IL-21. We furthermore demonstrated that ROCK2-mediated phosphorylation of IRF4 regulated the synthesis of IL-17 and IL-21 and the differentiation of Th17 cells. Whereas CD4+ T cells from WT mice activated ROCK2 physiologically under Th17 conditions, CD4+ T cells from 2 different mouse models of spontaneous autoimmunity aberrantly activated ROCK2 under neutral conditions. Moreover, administration of ROCK inhibitors ameliorated the deregulated production of IL-17 and IL-21 and the inflammatory and autoantibody responses observed in these autoimmune mice. Our findings thus uncover a crucial link among ROCK2, IRF4, and the production of IL-17 and IL-21 and support the idea that selective inhibition of ROCK2 could represent an important therapeutic regimen for the treatment of autoimmune disorders.

Figure 4

(A and B) ONP assay was performed on nuclear extracts from 293T cells transfected with empty vector or expression vector for WT IRF4 and (A) IRF4AA or (B) IRF4D446 or IRF4D447. Y27632 was added in selected cultures as indicated. Precipitated proteins were analyzed by Western blotting with IRF4 Ab; IRF4 levels in the input samples are shown below. (C and D) CD4+ T cells from Def6trap/trap single-KO (SKO) and Def6trap/trapIrf4–/– double-KO (DKO) mice were infected with control YFP-RV (V), WT IRF4, and IRF4 mutants-expressing retroviruses as indicated. Cells were harvested after 6 days, sorted for YFP+ cells, and restimulated with anti-CD3 and anti-CD28 for 48 hours in the presence or absence of 30 μM Y27632. Supernatants were then collected and assayed for IL-17 and IL-21 production by ELISA. *P ≤ 0.004. (E) CD4+ T cells purified from Def6+/+ and Def6trap/trap were stimulated as described in Figure 3. Nuclear extracts were analyzed by Western blotting using an Ab that recognizes pIRF4. The blot was later stripped and reprobed with an Ab against total IRF4. Lanes were run on the same gel but were noncontiguous (white lines). Data are representative of 3 independent experiments (A–E).